Hepatitis E virus gained notoriety as the cause of epidemics and sporadic cases of acute hepatitis in developing countries: examples include the 29,300 cases occurring during the New Delhi outbreak in 1956 and the 2,621 cases reported over 6 months in an Internally Displaced Persons Camp in Darfur in which pregnant women, as has been reported previously (1), had the highest mortality rate of 26-31% (2). HEV is the most, or second most, important cause of acute hepatitis in adults in developing countries. But contrary to recent dogma, the virus is not restricted to developing countries and sporadic cases are increasingly recognized in industrialized countries as awareness of the potential for infection spreads and tests for the virus are performed.
Historically, hepatitis E was described as an enterically-transmitted, self-limiting hepatitis that never progressed to chronicity (3). However, recently the first chronic hepatitis E was identified in Europe and chronicity has since been documented in immunocompromised solid-organ transplant recipients and HIV-infected individuals (4, 5, 6, 7). Although hepatitis E infection generally causes a mild to moderate disease, it occasionally has caused fulminant liver failure in acute cases, in chronically infected patients, and especially in those with underlying chronic liver disease or pregnancy (1, 2, 4, 5, 6, 7, 8). Additionally, hepatitis E has been misdiagnosed as drug-induced liver injury, thus complicating drug trials or treatment regimens (9). Since its discovery in 1983, documented HEV transmission was linked almost exclusively to contaminated water; that changed abruptly with the discovery of HEV infection following ingestion of uncooked deer meat (10, 11). Hepatitis E is now recognized as not just a waterborne-disease of developing countries but also as an emerging food-borne disease of industrialized countries (11, 12).
HEV is a small non-enveloped, single-stranded RNA virus with a genome size of 7.2 kb (3). The 7.2 kb genome of HEV is a single strand of positive sense RNA with three overlapping reading frames (ORFs). Approximately the first 5 kb serve as mRNA for the ORF1 polyprotein; it is not known if the polyprotein is proteolytically processed. ORF1 contains regions encoding methyl transferase/guanylyltransferase, NTPase/helicase, RNA-dependent RNA polymerase and ubiquinating activities. In addition, ORF1 encodes a Y region and X, or macro, region of unknown function and a hypervariable region (HVR) located near the middle of the ORF. The HVR varies in length and sequence among strains and genotypes: it tolerates small deletions but replication levels are severely depressed in cell culture. ORF2 and ORF3 are translated from a single bicistronic, subgenomic RNA to produce a 660 aa capsid protein and a 113 to 114 aa protein, respectively. The ORF3 protein is important for efficient release of virus particles from cultured cells and is required for infection of macaques.
To date, four HEV genotypes that infect humans are recognized (17). Genotype 1 and 2 infections have been identified exclusively in humans, whereas genotypes 3 and 4 viruses have been isolated from swine, deer, mongoose, cattle and rabbits in addition to humans (18). Genotypes 3 and 4 are ubiquitous in swine and undercooked pork may be a major source of zoonotic infections of humans (12, 18). However, cross-species transmission has not been extensively studied and additional zoonotic reservoirs probably exist.
HEV infection was long thought to be an acute infection lasting 2 to 7 weeks and that never progressed to chronicity. Recently, however, chronic HEV infection has been identified in immunesuppressed organ transplant patients or aids patients. Even more unexpectedly, some of these chronically ill patients have developed neurological symptoms and HEV has been isolated from cerebrospinal fluid. These chronic cases have been identified as genotype 3 infections.
HEV usually replicates to low titers in vivo and it has been exceedingly difficult to grow it in cultured cells and much of the virus life cycle is unknown. Okamoto and colleagues recently adapted a genotype 3 and a genotype 4 strain to replicate to high titers in two human cell lines, A549 lung cells and PLC/PRF/5 hepatoma cells (19, 20).
The epidemiology of HEV is far from understood and, in particular, the zoonotic aspects require further study. There is a need, therefore to develop HEV genotype strains that can replicate in cell culture. Further, there is a need to develop HEV vaccines, e.g., vaccines for genotype 3 strains.
The present invention relates, in part, to the discovery of a genotype 3 virus isolated from a chronically-infected patient (5) that was adapted to grow in human hepatoma cells and used to identify a set of human, swine and deer cell cultures permissive for HEV infection. The invention additionally relates to the characterization of the adapted virus to identify sequence changes that provide the ability to replicate in cell culture.